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• W2019012009 abstract "Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1–90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179–384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179–384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell. Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1–90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179–384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179–384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell. Hsp40 proteins make up an essential family of molecular chaperones that function to specify the cellular processes facilitated by Hsp70 (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 2Caplan A.J. Cyr D.M. Douglas M.G. Mol. Biol. Cell. 1993; 4: 555-563Crossref PubMed Scopus (194) Google Scholar, 3Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Silver P.A. Way J.C. Cell. 1993; 74: 5-6Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 5Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3130) Google Scholar). Prokaryotic and eukaryotic cells contain multiple Hsp40 proteins that act in specific pairs with Hsp70 at different cellular locations via conserved mechanisms (6Brodsky J.L. Hamamoto S. Feldheim D. Schekman R. J. Cell Biol. 1993; 120: 95-102Crossref PubMed Scopus (130) Google Scholar, 7Cyr D.M. Douglas M.G. J. Biol. Chem. 1994; 269: 9798-9804Abstract Full Text PDF PubMed Google Scholar, 8Schlenstedt G. Harris S. Risse B. Lill R. Silver P.A. J. Cell Biol. 1995; 129(4): 979-988Crossref Scopus (134) Google Scholar, 9Cyr D.M. Gething M.-J. Guidebook to Molecular Chaperones and Protein Folding Factors. Oxford University Press, Oxford, United Kingdom1997: 89-95Google Scholar). Escherichia coliDnaJ, the progenitor of the Hsp40 family, functions to promote protein folding, degradation of misfolded proteins, and bacteriophage DNA replication (3Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 10Zylicz M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1993; 339: 271-278Crossref PubMed Scopus (46) Google Scholar, 11Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). The mammalian Hsp40 protein Auxillin directs Hsp70 to uncoat clathrin-coated vesicles (12Barouch W. Prasad K. Greene L. Eisenberg E. Biochemistry. 1997; 36: 4303-4308Crossref PubMed Scopus (58) Google Scholar, 13Jiang R.-F. Greener T. Barouch W. Greene L. Eisenberg E. J. Biol. Chem. 1997; 272: 6141-6145Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The cysteine string protein of Drosophilia is palmitolyated and localized to neurosecretory vesicles, where it is proposed to direct Hsp70 to catalyze putative protein assembly/disassembly events required for neurotransmitter release (14Braun J.E.A. Wilbanks S.M. Scheller R.H. J. Biol. Chem. 1996; 271: 25989-25993Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 15Chamberlain L.H. Burgoyne R.D. Biochem. J. 1997; 322: 853-858Crossref PubMed Scopus (106) Google Scholar). The yeast protein Tim44 is a component of the import machinery that directs the mitochondrial cognate of Hsp70 to drive protein translocation into the matrix space (16Ungermann C. Neupert W. Cyr D.M. Science. 1994; 266: 1250-1253Crossref PubMed Scopus (225) Google Scholar, 17Ungermann C. Guiard B. Neupert W. Cyr D.M. EMBO J. 1996; 15: 735-744Crossref PubMed Scopus (90) Google Scholar, 18Cyr D.M. J. Bioenerg. Biomembr. 1997; 29: 29-34Crossref PubMed Scopus (7) Google Scholar, 19Lill R. Nargang F.E. Neupert W. Curr. Opin. Cell Biol. 1996; 8: 505-512Crossref PubMed Scopus (65) Google Scholar, 20Schatz G. J. Biol. Chem. 1996; 271: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Ydj1 is a cytosolic yeast protein that functions to deliver preproteins to organelle membranes (21Atencio D.P. Yaffe M.P. Mol. Cell. Biol. 1992; 12: 283-291Crossref PubMed Scopus (122) Google Scholar, 22Caplan A.J. Cyr D.M. Douglas M.G. Cell. 1992; 71: 1143-1155Abstract Full Text PDF PubMed Scopus (219) Google Scholar), refold model protein substrates (23Caplan A.J. Langley E. Wilson E.M. Vidal J. J. Biol. Chem. 1995; 270: 5251-5257Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24Kimura Y. Yahara I. Lindquist S. Science. 1995; 268: 1362-1365Crossref PubMed Scopus (219) Google Scholar), and facilitate the ubiquitin-dependent degradation of proteins (25Lee D.H. Sherman M.Y. Goldberg A.L. Mol. Cell. Biol. 1996; 16: 4773-4781Crossref PubMed Scopus (125) Google Scholar). Hsp70 proteins bind and release unfolded polypeptides in an ATP-dependent reaction cycle, with the ADP-form of Hsp70 exhibiting a higher affinity for substrates than the ATP-form (26Palleros D.R. Welch W.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5719-5723Crossref PubMed Scopus (281) Google Scholar). It is proposed that Hsp40 helps stabilize Hsp70-polypeptide complexes through promoting the conversion of Hsp70-ATP to Hsp70-ADP (26Palleros D.R. Welch W.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5719-5723Crossref PubMed Scopus (281) Google Scholar, 27Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar, 28Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 29McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar). However, in some instances, Hsp40 proteins can promote the release of proteins that are bound to Hsp70 (7Cyr D.M. Douglas M.G. J. Biol. Chem. 1994; 269: 9798-9804Abstract Full Text PDF PubMed Google Scholar). Ydj1 and a subset of Hsp40 family members also recognize aspects of non-native protein structure and bind protein folding intermediates in a manner that facilitates subsequent interactions between them and Hsp70 (28Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 30Wickner S. Hoskins J. McKenney K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7903-7907Crossref PubMed Scopus (138) Google Scholar, 31Cyr D.M. FEBS Lett. 1995; 359: 129-132Crossref PubMed Scopus (115) Google Scholar, 32Wawrzynow A. Zylicz M. J. Biol. Chem. 1995; 270: 19300-19306Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Minami Y. Hohfeld J. Ohtsuka K. Hartl F.-U. J. Biol. Chem. 1996; 271: 19617-19624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). In the absence of Hsp40, complex formation between Hsp70 and protein folding intermediates can be inefficient, which leads to the aggregation of substrate proteins (28Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 31Cyr D.M. FEBS Lett. 1995; 359: 129-132Crossref PubMed Scopus (115) Google Scholar, 34Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (208) Google Scholar). Sequence analysis suggests that the regulatory and chaperone actions of Hsp40 family members are catalyzed by four different conserved sequence motifs that originate from DnaJ (3Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (201) Google Scholar). Hsp40 proteins contain regions termed the J-domain, G/F-rich region, zinc finger-like domain, and conserved carboxyl terminus (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 9Cyr D.M. Gething M.-J. Guidebook to Molecular Chaperones and Protein Folding Factors. Oxford University Press, Oxford, United Kingdom1997: 89-95Google Scholar). The J-domain corresponds to the amino-terminal 70 amino acid residues of DnaJ and is found in all Hsp40 proteins. The J-domain is responsible for regulation of the ATP hydrolytic cycle of Hsp70. The NMR structure of J-domain demonstrates that it contains four regions of α-helical conformation, with helices I and II lying in an anti-parallel orientation to helices III and IV (35Hill R.B. Flanagan J.M. Prestegard J.H. Biochemistry. 1995; 34: 5587-5596Crossref PubMed Scopus (69) Google Scholar, 36Qian Y.Q. Patel D. Hartl F.U. McColl D.J. J. Mol. Biol. 1996; 260: 224-235Crossref PubMed Scopus (139) Google Scholar, 37Szyperski T. Pellecchia M. Wall D. Georgopoulos C. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11343-11347Crossref PubMed Scopus (141) Google Scholar). The orientation of helices I and II to III and IV is dictated by a loop in the J-domain formed by an HPD tripeptide that is found in all Hsp40 family members. Mutations in the HPD motif and faces of helices II and III abrogate the ability of Hsp40 proteins to interact with Hsp70 and regulate its ATPase activity (38Feldheim D. Rothblatt J. Schekman R. Mol. Cell. Biol. 1992; 12: 3288-3296Crossref PubMed Scopus (194) Google Scholar, 39Tsai J. Douglas M.G. J. Biol. Chem. 1996; 271: 9347-9354Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 40Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1994; 269: 5446-5451Abstract Full Text PDF PubMed Google Scholar, 41Dey B. Caplan A.J. Boschelli F. Mol. Biol. Cell. 1996; 7: 91-100Crossref PubMed Scopus (58) Google Scholar). Functions of the G/F-rich region, the zinc finger-like region and the conserved carboxyl terminus of Hsp40 proteins are not clear. The G/F-rich region is thought to act as a flexible spacer between the J-domain and other regions in Hsp40 proteins (4Silver P.A. Way J.C. Cell. 1993; 74: 5-6Abstract Full Text PDF PubMed Scopus (189) Google Scholar). In the case of DnaJ, the G/F-rich region is required in conjunction with the J-domain in order for productive interactions with E. coli Hsp70 to occur (42Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 43Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1995; 270: 2139-2144Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). However, because several Hsp40 family members lack the G/F-rich region and still interact with Hsp70, this conserved motif is not required for the function of all Hsp40 proteins (13Jiang R.-F. Greener T. Barouch W. Greene L. Eisenberg E. J. Biol. Chem. 1997; 272: 6141-6145Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 14Braun J.E.A. Wilbanks S.M. Scheller R.H. J. Biol. Chem. 1996; 271: 25989-25993Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cysteine-rich repeats that resemble C4 zinc binding domains comprise the zinc finger-like domain and play a role in polypeptide binding by Hsp40 family members (44Banecki B. Liberek K. Wall D. Wawrzynow A. Georgopoulos C. Bertoli E. Tanfani F. Zylicz M. J. Biol. Chem. 1996; 271: 14840-14848Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 45Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (276) Google Scholar). Fragments of DnaJ that contain the zinc finger-like region are capable of suppressing protein aggregation (45Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (276) Google Scholar). However, family members that lack the zinc finger-like region can form complexes with cellular proteins and appear to function as molecular chaperones (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 2Caplan A.J. Cyr D.M. Douglas M.G. Mol. Biol. Cell. 1993; 4: 555-563Crossref PubMed Scopus (194) Google Scholar, 3Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (201) Google Scholar). These data suggest that other regions of Hsp40 are also involved in interactions with non-native proteins. The conserved region in the carboxyl terminus of DnaJ lies adjacent to the zinc finger-like region and is typically present in Hsp40 proteins that bind polypeptides (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 2Caplan A.J. Cyr D.M. Douglas M.G. Mol. Biol. Cell. 1993; 4: 555-563Crossref PubMed Scopus (194) Google Scholar, 3Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 4Silver P.A. Way J.C. Cell. 1993; 74: 5-6Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 5Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3130) Google Scholar). However, a role for this domain in the function of Hsp40 family members has not been determined. To further define the mechanism of action for Hsp40 proteins in cellular protein metabolism, we have carried out a structure-function analysis of Ydj1. The functional analysis of protease-resistant fragments and mutant proteins has identified subdomains in Ydj1 responsible for its regulatory and chaperone activities. The J-domain functions independent of domains responsible for chaperone functions and is sufficient for regulation of Hsp70 ATPase activity. The conserved carboxyl terminus was identified as a region in Ydj1 that plays a major role in binding unfolded polypeptides. The zinc finger-like region was found to function in protein folding, but was not observed to be essential for stable binding of denatured model substrates. These data demonstrate that Ydj1 contains independent functional units that act via a coordinated mechanism to assist Hsp70 in interactions with cellular proteins. Hsp70 Ssa1 protein, termed Hsp70 for the remainder of the text, was overexpressed in yeast strain MW141 and purified by ATP-agarose, DEAE, and hydroxyapatite chromatography by established techniques (46Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). The open reading frames for Ydj1, Ydj1 H34Q (39Tsai J. Douglas M.G. J. Biol. Chem. 1996; 271: 9347-9354Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and Ydj1 G315D (24Kimura Y. Yahara I. Lindquist S. Science. 1995; 268: 1362-1365Crossref PubMed Scopus (219) Google Scholar) were subcloned into pET9d, overexpressed in E. coli, and purified by ion-exchange and hydroxyapatite chromatography as described previously (46Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Ydj1 C159T was tagged with six histidine residues at its amino terminus and purified by metal chelate chromatography (21Atencio D.P. Yaffe M.P. Mol. Cell. Biol. 1992; 12: 283-291Crossref PubMed Scopus (122) Google Scholar). In experiments with Ydj1 C159T, a histidine-tagged version of Ydj1 was utilized as a control. Ydj1 H34Q and Ydj1 G315D both behaved similarly to Ydj1 on ion-exchange columns, and these proteins all ran as dimers on gel filtration columns (data not shown). Therefore, it does not appear that the mutant forms of Ydj1 we have purified are grossly misfolded. Histidine-tagged Ydj1 was capable of suppressing the temperature sensitive growth phenotype of ΔYdj1 strains and appeared to function with the same efficiency as Ydj1 (data not shown). Additionally, purified His-Ydj1 behaved identically to the nontagged version in assays for regulatory and chaperone function described below (data not shown). Ydj1 C159T behaved similarly to His-tagged Ydj1 in all purification steps and ran as a dimer on gel filtration columns. Ydj1 (0.5 mg/ml) was digested with trypsin or proteinase K (PK) 1The abbreviations used are: PK, proteinase K; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis. at the concentrations indicated in digestion buffer (150 mm KCl, 25 mm Hepes, pH 7.4, and 10 mm DTT) for 1 h at 25 °C. Digestions were terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mm. Protease-resistant fragments of Ydj1 liberated were then analyzed by SDS-PAGE on 12.5 or 16% acrylamide gels for the trypsin and PK digestions, respectively. Digested Ydj1 (4.0 μg) was run out on SDS-PAGE gels to resolve the products of the proteolytic digestion from each other. Gels were then soaked for 20 min in transfer buffer (10 mm3-cyclohexylamino-1-propanesulfonic acid made in 10% methanol). Fragments were then electrophoretically transferred to polyvinylidene fluoride membranes in a Bio-Rad Mini-Wet cell blotter for 60 min with the power supply set at 50 V constant voltage. Membranes were then soaked in water for 5 min to remove the transfer buffer and stained with Coomassie Blue R-250 to illuminate the positions of the transferred protein fragments. Fragments were excised from the membranes, and the identity of the first six amino acid residues in them were determined with a Perkin-Elmer Applied Biosystems Microsequencing Apparatus. The molecular weights of the products liberated from Ydj1 by limited proteolysis were determined on a Perseptive Biosystems (Framingham, MA) Voyager Elite MALDI-TOF mass spectrometer. Aliquots of digested material (5 μl) were mixed with a saturated solution of α-cyano-4-hydroxy-cinnamic acid in a water:acetonitrile (50:50) mixture acidified with 0.1% trifluoroacetic acid. A 1-μl aliquot of this mixture was spotted onto a gold plate target. Ionization of the sample was then carried out with a nitrogen laser operating at 337 nm. A delayed extraction method was used in the determination of molecular weight. Measurement of ion flight times through the drift region of the mass spectrometer were carried out with a Tektronix (Beaverton, OR) TDS784A oscilloscope. The instrument was calibrated with external molecular weight standards. To analyze the function of Ydj1 (1–383), Ydj1 was digested with 2 μg/ml of trypsin for 30 min at 25 °C. The trypsin was then inactivated with 0.5 mmphenylmethylsulfonyl fluoride, and the activity of Ydj1 (1–383) was analyzed. At this trypsin concentration, Ydj1 is almost completely converted to Ydj1 (1–383) and contains less than 1% contamination with Ydj1 or other fragments (Fig. 1 B, lane 3). The levels of contaminants in this preparation were calculated to be insufficient to account for the observed activity of Ydj1 (1–383). To generate Ydj1 (102–394), Ydj1 (179–384), and Ydj1 (1–90) for functional analysis, digestions of Ydj1 with two different concentrations of PK were carried out. Then, the different Ydj1 fragments generated were separated from each other by high performance liquid chromatography gel filtration chromatography, concentrated, and immediately assayed for activity. To generate Ydj1 (102–394) virtually free of Ydj1 (179–384) and Ydj1 (1–90), 1 mg of Ydj1 (1 mg/ml) was digested with 1–2.0 μg/ml PK for 45 min at 25 °C. The concentration of PK utilized for this digestion was adjusted so that Ydj1 was efficiently converted to Ydj1 (102–394), but only minimal conversion to Ydj1 (179–384) could occur (see Fig. 1 C, lane 3). To separate Ydj1 (102–394) from Ydj1 (1–90) generated in protease digestions, reaction mixtures loaded onto a Bio-Rad Bio-Select G-250 gel filtration column. The column was then developed with a mobile phase consisting of 150 mm KCl, 20 mmHepes, pH 7.4, and 2 mm DTT that was pumped at 0.6 ml/min with a back pressure of 850 psi. Fractions (0.5 ml) were collected with a Bio-Rad Biologic medium pressure chromatography system and analyzed by SDS-PAGE to identify peaks. Ydj1 (102–394) and Ydj1 (179–384) eluted at 7.8 ml, and Ydj1 (1–90) eluted at 9.0 ml. The recovery of Ydj1 fragments in this step was typically 40% of the starting material. Gel filtration typically resolved the majority of Ydj1 (1–90) from Ydj1 (102–394), but a 3% contamination of this fragment remained, and Ydj1 (179–384) was present as a 5% contaminant. The quantity of Ydj1 (179–384) and Ydj1 (1–90) present in the Ydj1 (102–394) preparations were calculated to be insufficient to account for the activity of this fragments in functional assays. To prepare Ydj1 (179–394) free of Ydj1 (102–394), Ydj1 was digested under the conditions listed above, with 7.5 μg/ml PK. Ydj1 fragments liberated in a representative digestion are exhibited in Fig. 1 C, lane 5. To separate Ydj1 (179–384) from Ydj1 (1–90) and the unidentified lower molecular weight fragments generated, these samples were loaded onto on a Bio-Rad Bio-Select G-250 column and treated as described above. Ydj1 (179–384) eluted at 8.0 ml of mobile phase, and Ydj1 (1–90) eluted at 9.0 ml. The Ydj1 (179–384) fraction was routinely 93% pure with a 4% contamination of Ydj1 (1–90) and a 3% contamination of unidentified proteolytic products. The level of Ydj1 (1–90) and other contaminating bands present in this preparation were calculated to be insufficient to account for the activity attributed to Ydj1 (179–384). To generate a preparation of Ydj1 (1–90) that was free of larger Ydj1 fragments, digestions were carried out with 2.0 μg/ml as described above. Ydj1 (1–90) was then resolved from the Ydj1 (102–394) and Ydj1 (179–384) generated by the PK digestion (Fig. 1 C, lane 3) by gel filtration as described above. The Ydj1 (1–90) peak fraction was about 95% pure and contained a 5% contamination with Ydj1 (102–394) and Ydj1 (179–384). Ydj1 (1–90) ran as a broad band on SDS-PAGE gels, but microsequence analysis indicated that the majority of protein in this band had an amino terminus that corresponded to residues 1–6 of Ydj1 (Fig. 1 D) and to the J-domain of Ydj1. Analysis of the peptides in the Ydj1 band by mass spectroscopy indicated that the major fragment of Ydj1 present in this band was 9596 Da in size and corresponded to Ydj1 (1–90). We have calculated that the levels of Ydj1 (102–394) and Ydj1 (179–384) that contaminate the Ydj1 (1–90) preparation are too low to account for the functional activity attributed to this fraction. Purified Hsp70 was incubated in a reaction mixture containing 10 mm Hepes, pH 7.4, 80 mm KCl, 10 mm DTT, 1 mmMgCl2, and 50 μm ATP ([α-32P]ATP, 2.0–3.0 × 105 cpm/pmol) for 10 min at 30 °C. Reactions were then placed on ice, and duplicate 2-μl aliquots were assayed for ADP formation by thin layer chromatography on PEI cellulose plates (46Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Spontaneous ADP formation was assayed and subtracted prior to calculations for rates of ATP hydrolysis. The kinetics of Hsp70 ATPase activity were linear for at least 20 min under these experimental conditions (46Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Rates of rhodanese aggregation were determined by light scattering as described previously (31Cyr D.M. FEBS Lett. 1995; 359: 129-132Crossref PubMed Scopus (115) Google Scholar). Briefly, bovine rhodanese (50 μm; Sigma) was denatured for 1 h at 25 °C in 6 m guanidinium-HCl buffered with 20 mm Hepes, pH 7.4, and fresh 10 mm DTT. Denatured rhodanese was diluted 100-fold into 300 μl of reaction buffer composed of 20 mm Hepes, pH 7.4, 80 mmKCl, and 10 mm DTT. When present, respective chaperone proteins were added prior to rhodanese. Rates of rhodanese aggregation were determined by monitoring increases in light scattering over time with a spectrophotometer set at 320 nm at 25 °C. Refolding of firefly luciferase (Promega) by Ydj1 and Hsp70 was carried out as described previously (47Levy E.J. McCarty J. Bukau B. Chirico W.J. FEBS Lett. 1995; 368: 435-440Crossref PubMed Scopus (68) Google Scholar). Luciferase (13 mg/ml) was diluted 42-fold into denaturation buffer (25 mm Hepes, pH 7.4, 50 mm KCl, 5 mm MgCl2, 6 m guanidinum-HCl, 5 mm DTT). The denaturation reaction was allowed to proceed for 40 min at 25 °C, and then a 1-μl aliquot was removed and mixed with 125 μl of refolding buffer (25 mm Hepes, pH 7.4, 50 mm KCl, 5 mm MgCl2, 1 mm ATP) and incubated at 30 °C. Aliquots of 1 μl were removed from the refolding buffer at indicated times and mixed with 60 μl of luciferase assay reagent (Promega). Luciferase activity was then measured with a Turner TD-20/20 luminometer. Ydj1 (1.6 μm), the Ydj1 mutants (1.6 μm), and Hsp70 (0.8 μm) were added to reactions prior to luciferase. The level of luciferase activity observed when Ydj1 and Hsp70 were present in reaction mixture was 17-fold higher than that activity observed when these chaperone proteins were omitted, which is consistent with previous reports (47Levy E.J. McCarty J. Bukau B. Chirico W.J. FEBS Lett. 1995; 368: 435-440Crossref PubMed Scopus (68) Google Scholar). Firefly luciferase (7.5 μl) in a 100 μm stock that was denatured in 6 mguanidinium-HCl, 10 mm Hepes, pH 7.4, and 10 mmDTT and was mixed with 292.5 μl of folding buffer (150 mmKCl, 10 mm Hepes, pH 7.4, 1 mm DTT) to give a final concentration of 2.5 μm. Where indicated, 5 μm Ydj1 or its mutant forms were added to folding buffer prior to the addition of luciferase. After a 20-min incubation period at 25 °C to allow complexes between Ydj1 and luciferase to form, reactions were loaded onto a Novarose SE-1000/17 column (Bio-Rad) equilibrated with folding buffer and eluted at a flow rate of 1 ml/min. To determine where Ydj1 and luciferase migrated, 0.5 ml fractions were collected, and the proteins present in 400 μl aliquots of each fraction were precipitated by the addition of 1.2 ml of acetone. Pelleted material was resuspended in 10 μl of sample buffer and analyzed on 10% SDS-PAGE gels. The concentration of Ydj1 and luciferase present in different fractions was quantitated by densitometry. In the absence of luciferase, Ydj1 eluted at 9.0 ml of mobile phase. In the absence of Ydj1, less than 1% of the unfolded luciferase eluted, whereas almost 50% of native luciferase was recovered in a peak at 10 ml. When Ydj1 (5 μm) and native luciferase (2.5 μm) were mixed and then injected onto the column, no alteration in the migration of either protein was observed. When Ydj1 and unfolded lucif" @default.
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